Assessment of the validity of the double superhelix model for reconstituted high density lipoproteins: a combined computational-experimental approach

Abstract

For several decades, the standard model for high density lipoprotein (HDL) particles reconstituted from apolipoprotein A-I (apoA-I) and phospholipid (apoA-I/HDL) has been a discoidal particle ∼100 Å in diameter and the thickness of a phospholipid bilayer. Recently, Wu et al. (Wu, Z., Gogonea, V., Lee, X., Wagner, M. A., Li, X. M., Huang, Y., Undurti, A., May, R. P., Haertlein, M., Moulin, M., Gutsche, I., Zaccai, G., Didonato, J. A., and Hazen, S. L. (2009) J. Biol. Chem. 284, 36605-36619) used small angle neutron scattering to develop a new model they termed double superhelix (DSH) apoA-I that is dramatically different from the standard model. Their model possesses an open helical shape that wraps around a prolate ellipsoidal type I hexagonal lyotropic liquid crystalline phase. Here, we used three independent approaches, molecular dynamics, EM tomography, and fluorescence resonance energy transfer spectroscopy (FRET) to assess the validity of the DSH model. (i) By using molecular dynamics, two different approaches, all-atom simulated annealing and coarse-grained simulation, show that initial ellipsoidal DSH particles rapidly collapse to discoidal bilayer structures. These results suggest that, compatible with current knowledge of lipid phase diagrams, apoA-I cannot stabilize hexagonal I phase particles of phospholipid. (ii) By using EM, two different approaches, negative stain and cryo-EM tomography, show that reconstituted apoA-I/HDL particles are discoidal in shape. (iii) By using FRET, reconstituted apoA-I/HDL particles show a 28-34-Å intermolecular separation between terminal domain residues 40 and 240, a distance that is incompatible with the dimensions of the DSH model. Therefore, we suggest that, although novel, the DSH model is energetically unfavorable and not likely to be correct. Rather, we conclude that all evidence supports the likelihood that reconstituted apoA-I/HDL particles, in general, are discoidal in shape.

FIGURE 1.

Cross-eyed stereo images of the starting all-atom and coarse-grained DSH model made with Rasmol. Color code used is as follows. Protein: G* domain (residues 1–43), white; helix 5, green; helix 8, cyan; helix 10, red; the remainder of protein is slate blue; prolines, yellow. POPC: acyl chains, black; P, gold; cholines, sky blue. UC, magenta. The left-hand images are rotated 180° to create the right-hand images. A, starting DSH model for nascent HDL used in MDSA simulations. B, protein alone; the distance between the N and C termini is indicated by the black bar. C, starting DSH model in A that was coarse-grained and used in CGMD simulations. D, protein alone; the distance between the N and C termini is indicated by the black bar.

FIGURE 2.

r.m.s.d. plots for the three MD simulations. A, r.m.s.d. versus time for the two consecutive MDSA simulations of the starting all-atom DSH model. B, r.m.s.d. versus time for the CGMD simulation of the starting CG DSH model. C, r.m.s.d. versus time for the MDSA simulation of the starting discoidal model containing 200 POPC, 20 UC, and 2 apoA-I.

FIGURE 3.

Space-filling models made with VMD using depth-cueing viewed from the lipid-rich side showing intermediates in the collapse of the starting DSH structure to a discoidal shape during MDSA and CGMD simulations. Color code, same as for Fig. 1. A, structure of MDSA simulation at 0, 2, 4, 5, and 60 ns. A-1, Entire particle. A-2, protein only. r.m.s.d. for protein in each structure is shown in parentheses. B, structure of CGMD simulation at 0, 0.16, 0.32, 0.64, and 60 μs equivalents. B-1, entire particle. B-2, protein only. r.m.s.d. for protein in each structure is shown in parentheses.

FIGURE 4.

Final structures of all-atom and coarse-grained MD simulations of the starting DSH model compared with an all-atom 30-ns MD simulation of a discoidal model containing 200 POPC, 20 UC, and 2 apoA-I. All images were made with Rasmol. Color code, same as for Fig. 1. Each structure is shown from the top and side in cross-eyed stereo and as a nonstereo cross-sectional view of each structure from the side. A, MDSA of the starting all-atom model simulated for 60 ns. B, CGMD simulation of the starting coarse-grained DSH model simulated for 60 μs. C, all-atom 30-ns MD simulation of a discoidal model containing 200 POPC, 20 UC, and 2 apoA-I.

FIGURE 5.

Discoidal shapes of reconstituted apoA-I/HDL particles revealed by negative stain and electron cryo-tomography. In each view the axis of tilt is vertical to the images (diagrammatically illustrated in upper and bottom center of A). Selected titled images are linked by dotted arrows. Relative tilt angles are indicated in each image. Scale bars, 200 Å. A, three selected tilted views of apoA-I/HDL particles from one negative stain electron microscopic field. B–G, selected tilted views of near native state apoA-I/HDL particles embedded in vitreous ice from six cryo-electron microscopic fields.

FIGURE 6.

Intramolecular FRET between positions 40 and 240. Fluorescence emission (excitation wavelength 280 nm) of W@40 and W@40:L240C-AE was observed at 310–560 nm. Sample concentrations were 0.1 mg/ml. The rHDL samples W@40 and W@40:L240C-AE consisted of W@40 and AEDANS-labeled apoA-I (W@40:L240C-AE) in a 1:5 ratio with W@, respectively. The W@40 and W@40:L240C-AE rHDL spectra were normalized, correcting for their proportion (1:5 with W@) in rHDL. Background fluorescence was eliminated by subtracting the emission spectrum of rHDL consisting of W@ from the normalized emission spectrum of W@40 and W@40:L240C-AE rHDL. Energy transfer efficiency (E) was calculated from background corrected spectra by comparing Trp fluorescence intensities (integrated over 310–425 nm) of W@40 (donor only) and W@40:L240C-AE (donor-acceptor).

FIGURE 7.

Cross-eyed stereo image of the Cα alignment of the protein conformation of the final structure of the MD simulation by Hazen and co-workers (26) with the stereo image of our 60-ns MDSA simulation of the starting DSH model made with VMD. The Hazen and co-workers (26) model is in magenta, and our 60 ns MDSA simulation of the starting DSH model (11) is in green. N- and C-terminal residues are space-filling blue and red, respectively.

FIGURE 8.

Cross-eyed stereo images made with Rasmol showing the collapse of the central cavity in both the initial DSH model and the recently published 60-ns MD simulation of the DSH model (26) following MD simulations using the NPT ensemble. A, cross-sectional image of the initial DSH model (11). B, cross-sectional image of the starting DSH model after a 2-ns MD simulation at 310 using the NPT ensemble. C, cross-sectional image of the 60-ns MD simulation of the DSH model showing the large central cavity. D, cross-sectional image of the 60-ns MD simulation of the DSH model after 10 ns of MD simulation at 310 using the NPT ensemble. Protein is gray; POPC acyl chains are black except for terminal methyls that are green; POPC head groups are blue, red, and gold; UC is black.